Specify the tissue of interest, run the boilerplate code which sets up the functions and environment, load the tissue object.

tissue_of_interest = "Tongue"
library(here)
here() starts at /Users/Jintonic/Documents/GitHub/tabula-muris
source(here("00_data_ingest", "02_tissue_analysis_rmd", "boilerplate.R"))

Attaching package: ‘dplyr’

The following object is masked from ‘package:Biobase’:

    combine

The following objects are masked from ‘package:BiocGenerics’:

    combine, intersect, setdiff, union

The following objects are masked from ‘package:stats’:

    filter, lag

The following objects are masked from ‘package:base’:

    intersect, setdiff, setequal, union
tiss = load_tissue_droplet(tissue_of_interest)
Performing log-normalization
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
[1] "Scaling data matrix"

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Calculating gene means
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variance to mean ratios
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|

Later on (in FindClusters and TSNE) you will pick a number of principal components to use. This has the effect of keeping the major directions of variation in the data and, ideally, supressing noise. There is no correct answer to the number to use, but a decent rule of thumb is to go until the plot plateaus.

PCElbowPlot(object = tiss)

Choose the number of principal components to use.

# Set number of principal components. 
n.pcs = 14

The clustering is performed based on a nearest neighbors graph. Cells that have similar expression will be joined together. The Louvain algorithm looks for groups of cells with high modularity–more connections within the group than between groups. The resolution parameter determines the scale…higher resolution will give more clusters, lower resolution will give fewer.

For the top-level clustering, aim to under-cluster instead of over-cluster. It will be easy to subset groups and further analyze them below.

# Set resolution 
res.used <- 0.4
tiss <- FindClusters(object = tiss, reduction.type = "pca", dims.use = 1:n.pcs, 
    resolution = res.used, print.output = 0, save.SNN = TRUE, force.recalc = TRUE)

To visualize

# If cells are too spread out, you can raise the perplexity. If you have few cells, try a lower perplexity (but never less than 10).
tiss <- RunTSNE(object = tiss, dims.use = 1:n.pcs, seed.use = 20, perplexity=35, dim.embed = 2)
TSNEPlot(object = tiss, do.label = T, pt.size = 1.2, label.size = 4)

Check expression of genes of interset.

Dotplots let you see the intensity of exppression and the fraction of cells expressing for each of your genes of interest.

How big are the clusters?

table(tiss@ident)

   0    1    2    3    4    5    6    7    8    9   10 
1389 1095 1079  869  815  602  601  485  366  199   38 
tiss1=BuildClusterTree(tiss)
[1] "Finished averaging RNA for cluster 0"
[1] "Finished averaging RNA for cluster 1"
[1] "Finished averaging RNA for cluster 2"
[1] "Finished averaging RNA for cluster 3"
[1] "Finished averaging RNA for cluster 4"
[1] "Finished averaging RNA for cluster 5"
[1] "Finished averaging RNA for cluster 6"
[1] "Finished averaging RNA for cluster 7"
[1] "Finished averaging RNA for cluster 8"
[1] "Finished averaging RNA for cluster 9"
[1] "Finished averaging RNA for cluster 10"

#cluster1.markers <- FindMarkers(object = tiss, ident.1 = 1, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster2.markers <- FindMarkers(object = tiss, ident.1 = 2, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster3.markers <- FindMarkers(object = tiss, ident.1 = 3, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster4.markers <- FindMarkers(object = tiss, ident.1 = 4, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster5.markers <- FindMarkers(object = tiss, ident.1 = 5, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
cluster6.markers <- FindMarkers(object = tiss, ident.1 = 6, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster7.markers <- FindMarkers(object = tiss, ident.1 = 7, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster8.markers <- FindMarkers(object = tiss, ident.1 = 8, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster9.markers <- FindMarkers(object = tiss, ident.1 = 9, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster0.markers <- FindMarkers(object = tiss, ident.1 = 0, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)

Which markers identify a specific cluster?

print(x = head(x = cluster6.markers, n = 30))

You can also compute all markers for all clusters at once. This may take some time.

tiss.markers <- FindAllMarkers(object = tiss, only.pos = TRUE, min.pct = 0.25, thresh.use = 0.25)

   |                                                  | 0 % ~calculating  
   |+                                                 | 1 % ~02m 53s      
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   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 47s

   |                                                  | 0 % ~calculating  
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   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 57s

   |                                                  | 0 % ~calculating  
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   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 02m 36s

   |                                                  | 0 % ~calculating  
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   |+++++++++++++++++++++++++++++++++++++++           | 77% ~21s          
   |++++++++++++++++++++++++++++++++++++++++          | 78% ~20s          
   |++++++++++++++++++++++++++++++++++++++++          | 79% ~19s          
   |+++++++++++++++++++++++++++++++++++++++++         | 80% ~18s          
   |+++++++++++++++++++++++++++++++++++++++++         | 81% ~17s          
   |++++++++++++++++++++++++++++++++++++++++++        | 82% ~16s          
   |++++++++++++++++++++++++++++++++++++++++++        | 84% ~15s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~14s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 86% ~13s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~12s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 88% ~11s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~11s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~10s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~09s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~08s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~07s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~06s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~05s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~04s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~03s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~02s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~01s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 32s

   |                                                  | 0 % ~calculating  
   |+                                                 | 1 % ~03m 05s      
   |++                                                | 2 % ~03m 06s      
   |++                                                | 3 % ~03m 13s      
   |+++                                               | 4 % ~03m 13s      
   |+++                                               | 5 % ~03m 12s      
   |++++                                              | 6 % ~03m 19s      
   |++++                                              | 7 % ~03m 20s      
   |+++++                                             | 8 % ~03m 49s      
   |+++++                                             | 9 % ~03m 46s      
   |++++++                                            | 10% ~03m 40s      
   |++++++                                            | 11% ~03m 33s      
   |+++++++                                           | 12% ~03m 27s      
   |+++++++                                           | 13% ~03m 23s      
   |++++++++                                          | 14% ~03m 19s      
   |++++++++                                          | 15% ~03m 14s      
   |+++++++++                                         | 16% ~03m 11s      
   |+++++++++                                         | 18% ~03m 08s      
   |++++++++++                                        | 19% ~03m 04s      
   |++++++++++                                        | 20% ~03m 01s      
   |+++++++++++                                       | 21% ~02m 58s      
   |+++++++++++                                       | 22% ~02m 55s      
   |++++++++++++                                      | 23% ~02m 52s      
   |++++++++++++                                      | 24% ~02m 50s      
   |+++++++++++++                                     | 25% ~02m 49s      
   |+++++++++++++                                     | 26% ~02m 46s      
   |++++++++++++++                                    | 27% ~02m 44s      
   |++++++++++++++                                    | 28% ~02m 41s      
   |+++++++++++++++                                   | 29% ~02m 38s      
   |+++++++++++++++                                   | 30% ~02m 38s      
   |++++++++++++++++                                  | 31% ~02m 36s      
   |++++++++++++++++                                  | 32% ~02m 35s      
   |+++++++++++++++++                                 | 33% ~02m 32s      
   |++++++++++++++++++                                | 34% ~02m 30s      
   |++++++++++++++++++                                | 35% ~02m 27s      
   |+++++++++++++++++++                               | 36% ~02m 24s      
   |+++++++++++++++++++                               | 37% ~02m 22s      
   |++++++++++++++++++++                              | 38% ~02m 19s      
   |++++++++++++++++++++                              | 39% ~02m 16s      
   |+++++++++++++++++++++                             | 40% ~02m 14s      
   |+++++++++++++++++++++                             | 41% ~02m 12s      
   |++++++++++++++++++++++                            | 42% ~02m 09s      
   |++++++++++++++++++++++                            | 43% ~02m 07s      
   |+++++++++++++++++++++++                           | 44% ~02m 05s      
   |+++++++++++++++++++++++                           | 45% ~02m 02s      
   |++++++++++++++++++++++++                          | 46% ~01m 60s      
   |++++++++++++++++++++++++                          | 47% ~01m 57s      
   |+++++++++++++++++++++++++                         | 48% ~01m 55s      
   |+++++++++++++++++++++++++                         | 49% ~01m 53s      
   |++++++++++++++++++++++++++                        | 51% ~01m 51s      
   |++++++++++++++++++++++++++                        | 52% ~01m 48s      
   |+++++++++++++++++++++++++++                       | 53% ~01m 46s      
   |+++++++++++++++++++++++++++                       | 54% ~01m 43s      
   |++++++++++++++++++++++++++++                      | 55% ~01m 41s      
   |++++++++++++++++++++++++++++                      | 56% ~01m 38s      
   |+++++++++++++++++++++++++++++                     | 57% ~01m 36s      
   |+++++++++++++++++++++++++++++                     | 58% ~01m 34s      
   |++++++++++++++++++++++++++++++                    | 59% ~01m 32s      
   |++++++++++++++++++++++++++++++                    | 60% ~01m 30s      
   |+++++++++++++++++++++++++++++++                   | 61% ~01m 28s      
   |+++++++++++++++++++++++++++++++                   | 62% ~01m 26s      
   |++++++++++++++++++++++++++++++++                  | 63% ~01m 23s      
   |++++++++++++++++++++++++++++++++                  | 64% ~01m 22s      
   |+++++++++++++++++++++++++++++++++                 | 65% ~01m 19s      
   |+++++++++++++++++++++++++++++++++                 | 66% ~01m 17s      
   |++++++++++++++++++++++++++++++++++                | 67% ~01m 15s      
   |+++++++++++++++++++++++++++++++++++               | 68% ~01m 13s      
   |+++++++++++++++++++++++++++++++++++               | 69% ~01m 11s      
   |++++++++++++++++++++++++++++++++++++              | 70% ~01m 08s      
   |++++++++++++++++++++++++++++++++++++              | 71% ~01m 06s      
   |+++++++++++++++++++++++++++++++++++++             | 72% ~01m 04s      
   |+++++++++++++++++++++++++++++++++++++             | 73% ~01m 01s      
   |++++++++++++++++++++++++++++++++++++++            | 74% ~59s          
   |++++++++++++++++++++++++++++++++++++++            | 75% ~56s          
   |+++++++++++++++++++++++++++++++++++++++           | 76% ~54s          
   |+++++++++++++++++++++++++++++++++++++++           | 77% ~52s          
   |++++++++++++++++++++++++++++++++++++++++          | 78% ~49s          
   |++++++++++++++++++++++++++++++++++++++++          | 79% ~47s          
   |+++++++++++++++++++++++++++++++++++++++++         | 80% ~45s          
   |+++++++++++++++++++++++++++++++++++++++++         | 81% ~43s          
   |++++++++++++++++++++++++++++++++++++++++++        | 82% ~40s          
   |++++++++++++++++++++++++++++++++++++++++++        | 84% ~38s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~35s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 86% ~33s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~31s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 88% ~28s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~26s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~24s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~21s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~19s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~16s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~14s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~12s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~09s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~07s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~05s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~02s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 03m 46s

   |                                                  | 0 % ~calculating  
   |+                                                 | 1 % ~01m 18s      
   |++                                                | 2 % ~01m 16s      
   |++                                                | 3 % ~01m 16s      
   |+++                                               | 5 % ~01m 13s      
   |+++                                               | 6 % ~01m 12s      
   |++++                                              | 7 % ~01m 11s      
   |+++++                                             | 8 % ~01m 11s      
   |+++++                                             | 9 % ~01m 09s      
   |++++++                                            | 10% ~01m 12s      
   |++++++                                            | 11% ~01m 12s      
   |+++++++                                           | 13% ~01m 11s      
   |+++++++                                           | 14% ~01m 11s      
   |++++++++                                          | 15% ~01m 12s      
   |+++++++++                                         | 16% ~01m 13s      
   |+++++++++                                         | 17% ~01m 12s      
   |++++++++++                                        | 18% ~01m 10s      
   |++++++++++                                        | 20% ~01m 09s      
   |+++++++++++                                       | 21% ~01m 08s      
   |+++++++++++                                       | 22% ~01m 06s      
   |++++++++++++                                      | 23% ~01m 05s      
   |+++++++++++++                                     | 24% ~01m 04s      
   |+++++++++++++                                     | 25% ~01m 03s      
   |++++++++++++++                                    | 26% ~01m 02s      
   |++++++++++++++                                    | 28% ~01m 02s      
   |+++++++++++++++                                   | 29% ~01m 01s      
   |+++++++++++++++                                   | 30% ~60s          
   |++++++++++++++++                                  | 31% ~58s          
   |+++++++++++++++++                                 | 32% ~57s          
   |+++++++++++++++++                                 | 33% ~56s          
   |++++++++++++++++++                                | 34% ~55s          
   |++++++++++++++++++                                | 36% ~54s          
   |+++++++++++++++++++                               | 37% ~53s          
   |+++++++++++++++++++                               | 38% ~52s          
   |++++++++++++++++++++                              | 39% ~51s          
   |+++++++++++++++++++++                             | 40% ~50s          
   |+++++++++++++++++++++                             | 41% ~49s          
   |++++++++++++++++++++++                            | 43% ~48s          
   |++++++++++++++++++++++                            | 44% ~47s          
   |+++++++++++++++++++++++                           | 45% ~46s          
   |+++++++++++++++++++++++                           | 46% ~45s          
   |++++++++++++++++++++++++                          | 47% ~44s          
   |+++++++++++++++++++++++++                         | 48% ~43s          
   |+++++++++++++++++++++++++                         | 49% ~42s          
   |++++++++++++++++++++++++++                        | 51% ~41s          
   |++++++++++++++++++++++++++                        | 52% ~40s          
   |+++++++++++++++++++++++++++                       | 53% ~39s          
   |++++++++++++++++++++++++++++                      | 54% ~38s          
   |++++++++++++++++++++++++++++                      | 55% ~37s          
   |+++++++++++++++++++++++++++++                     | 56% ~36s          
   |+++++++++++++++++++++++++++++                     | 57% ~35s          
   |++++++++++++++++++++++++++++++                    | 59% ~34s          
   |++++++++++++++++++++++++++++++                    | 60% ~33s          
   |+++++++++++++++++++++++++++++++                   | 61% ~32s          
   |++++++++++++++++++++++++++++++++                  | 62% ~31s          
   |++++++++++++++++++++++++++++++++                  | 63% ~30s          
   |+++++++++++++++++++++++++++++++++                 | 64% ~29s          
   |+++++++++++++++++++++++++++++++++                 | 66% ~28s          
   |++++++++++++++++++++++++++++++++++                | 67% ~27s          
   |++++++++++++++++++++++++++++++++++                | 68% ~26s          
   |+++++++++++++++++++++++++++++++++++               | 69% ~25s          
   |++++++++++++++++++++++++++++++++++++              | 70% ~24s          
   |++++++++++++++++++++++++++++++++++++              | 71% ~23s          
   |+++++++++++++++++++++++++++++++++++++             | 72% ~22s          
   |+++++++++++++++++++++++++++++++++++++             | 74% ~21s          
   |++++++++++++++++++++++++++++++++++++++            | 75% ~20s          
   |++++++++++++++++++++++++++++++++++++++            | 76% ~19s          
   |+++++++++++++++++++++++++++++++++++++++           | 77% ~19s          
   |++++++++++++++++++++++++++++++++++++++++          | 78% ~18s          
   |++++++++++++++++++++++++++++++++++++++++          | 79% ~17s          
   |+++++++++++++++++++++++++++++++++++++++++         | 80% ~16s          
   |+++++++++++++++++++++++++++++++++++++++++         | 82% ~15s          
   |++++++++++++++++++++++++++++++++++++++++++        | 83% ~14s          
   |++++++++++++++++++++++++++++++++++++++++++        | 84% ~13s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~12s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 86% ~11s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~10s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~09s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~08s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~07s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~06s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~05s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 94% ~05s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~04s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~03s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~02s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~01s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 19s

   |                                                  | 0 % ~calculating  
   |+                                                 | 1 % ~02m 41s      
   |++                                                | 2 % ~02m 39s      
   |++                                                | 3 % ~02m 36s      
   |+++                                               | 4 % ~02m 33s      
   |+++                                               | 5 % ~02m 30s      
   |++++                                              | 7 % ~02m 29s      
   |++++                                              | 8 % ~02m 26s      
   |+++++                                             | 9 % ~02m 29s      
   |+++++                                             | 10% ~02m 26s      
   |++++++                                            | 11% ~02m 24s      
   |++++++                                            | 12% ~02m 22s      
   |+++++++                                           | 13% ~02m 20s      
   |++++++++                                          | 14% ~02m 17s      
   |++++++++                                          | 15% ~02m 15s      
   |+++++++++                                         | 16% ~02m 13s      
   |+++++++++                                         | 17% ~02m 14s      
   |++++++++++                                        | 18% ~02m 12s      
   |++++++++++                                        | 20% ~02m 10s      
   |+++++++++++                                       | 21% ~02m 08s      
   |+++++++++++                                       | 22% ~02m 05s      
   |++++++++++++                                      | 23% ~02m 03s      
   |++++++++++++                                      | 24% ~02m 02s      
   |+++++++++++++                                     | 25% ~01m 60s      
   |++++++++++++++                                    | 26% ~01m 58s      
   |++++++++++++++                                    | 27% ~01m 56s      
   |+++++++++++++++                                   | 28% ~01m 54s      
   |+++++++++++++++                                   | 29% ~01m 52s      
   |++++++++++++++++                                  | 30% ~01m 50s      
   |++++++++++++++++                                  | 32% ~01m 50s      
   |+++++++++++++++++                                 | 33% ~01m 48s      
   |+++++++++++++++++                                 | 34% ~01m 46s      
   |++++++++++++++++++                                | 35% ~01m 44s      
   |++++++++++++++++++                                | 36% ~01m 43s      
   |+++++++++++++++++++                               | 37% ~01m 41s      
   |++++++++++++++++++++                              | 38% ~01m 39s      
   |++++++++++++++++++++                              | 39% ~01m 37s      
   |+++++++++++++++++++++                             | 40% ~01m 35s      
   |+++++++++++++++++++++                             | 41% ~01m 34s      
   |++++++++++++++++++++++                            | 42% ~01m 32s      
   |++++++++++++++++++++++                            | 43% ~01m 31s      
   |+++++++++++++++++++++++                           | 45% ~01m 29s      
   |+++++++++++++++++++++++                           | 46% ~01m 30s      
   |++++++++++++++++++++++++                          | 47% ~01m 32s      
   |++++++++++++++++++++++++                          | 48% ~01m 30s      
   |+++++++++++++++++++++++++                         | 49% ~01m 28s      
   |+++++++++++++++++++++++++                         | 50% ~01m 26s      
   |++++++++++++++++++++++++++                        | 51% ~01m 24s      
   |+++++++++++++++++++++++++++                       | 52% ~01m 22s      
   |+++++++++++++++++++++++++++                       | 53% ~01m 20s      
   |++++++++++++++++++++++++++++                      | 54% ~01m 18s      
   |++++++++++++++++++++++++++++                      | 55% ~01m 16s      
   |+++++++++++++++++++++++++++++                     | 57% ~01m 14s      
   |+++++++++++++++++++++++++++++                     | 58% ~01m 12s      
   |++++++++++++++++++++++++++++++                    | 59% ~01m 10s      
   |++++++++++++++++++++++++++++++                    | 60% ~01m 08s      
   |+++++++++++++++++++++++++++++++                   | 61% ~01m 07s      
   |+++++++++++++++++++++++++++++++                   | 62% ~01m 05s      
   |++++++++++++++++++++++++++++++++                  | 63% ~01m 03s      
   |+++++++++++++++++++++++++++++++++                 | 64% ~01m 01s      
   |+++++++++++++++++++++++++++++++++                 | 65% ~59s          
   |++++++++++++++++++++++++++++++++++                | 66% ~57s          
   |++++++++++++++++++++++++++++++++++                | 67% ~55s          
   |+++++++++++++++++++++++++++++++++++               | 68% ~53s          
   |+++++++++++++++++++++++++++++++++++               | 70% ~51s          
   |++++++++++++++++++++++++++++++++++++              | 71% ~50s          
   |++++++++++++++++++++++++++++++++++++              | 72% ~49s          
   |+++++++++++++++++++++++++++++++++++++             | 73% ~47s          
   |+++++++++++++++++++++++++++++++++++++             | 74% ~45s          
   |++++++++++++++++++++++++++++++++++++++            | 75% ~43s          
   |+++++++++++++++++++++++++++++++++++++++           | 76% ~41s          
   |+++++++++++++++++++++++++++++++++++++++           | 77% ~39s          
   |++++++++++++++++++++++++++++++++++++++++          | 78% ~37s          
   |++++++++++++++++++++++++++++++++++++++++          | 79% ~35s          
   |+++++++++++++++++++++++++++++++++++++++++         | 80% ~33s          
   |+++++++++++++++++++++++++++++++++++++++++         | 82% ~31s          
   |++++++++++++++++++++++++++++++++++++++++++        | 83% ~30s          
   |++++++++++++++++++++++++++++++++++++++++++        | 84% ~28s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~26s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 86% ~24s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~22s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 88% ~20s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~18s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 90% ~17s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~15s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 92% ~13s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~11s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~09s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~07s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~06s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~04s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~02s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 02m 49s

   |                                                  | 0 % ~calculating  
   |+                                                 | 1 % ~01m 06s      
   |++                                                | 2 % ~01m 07s      
   |++                                                | 4 % ~01m 06s      
   |+++                                               | 5 % ~01m 05s      
   |+++                                               | 6 % ~01m 04s      
   |++++                                              | 7 % ~01m 03s      
   |+++++                                             | 8 % ~01m 02s      
   |+++++                                             | 10% ~01m 01s      
   |++++++                                            | 11% ~01m 00s      
   |++++++                                            | 12% ~59s          
   |+++++++                                           | 13% ~59s          
   |++++++++                                          | 14% ~58s          
   |++++++++                                          | 15% ~58s          
   |+++++++++                                         | 17% ~57s          
   |+++++++++                                         | 18% ~56s          
   |++++++++++                                        | 19% ~55s          
   |+++++++++++                                       | 20% ~54s          
   |+++++++++++                                       | 21% ~53s          
   |++++++++++++                                      | 23% ~53s          
   |++++++++++++                                      | 24% ~53s          
   |+++++++++++++                                     | 25% ~52s          
   |++++++++++++++                                    | 26% ~51s          
   |++++++++++++++                                    | 27% ~50s          
   |+++++++++++++++                                   | 29% ~49s          
   |+++++++++++++++                                   | 30% ~49s          
   |++++++++++++++++                                  | 31% ~48s          
   |+++++++++++++++++                                 | 32% ~47s          
   |+++++++++++++++++                                 | 33% ~46s          
   |++++++++++++++++++                                | 35% ~45s          
   |++++++++++++++++++                                | 36% ~44s          
   |+++++++++++++++++++                               | 37% ~43s          
   |++++++++++++++++++++                              | 38% ~43s          
   |++++++++++++++++++++                              | 39% ~42s          
   |+++++++++++++++++++++                             | 40% ~41s          
   |+++++++++++++++++++++                             | 42% ~40s          
   |++++++++++++++++++++++                            | 43% ~39s          
   |+++++++++++++++++++++++                           | 44% ~38s          
   |+++++++++++++++++++++++                           | 45% ~38s          
   |++++++++++++++++++++++++                          | 46% ~37s          
   |++++++++++++++++++++++++                          | 48% ~36s          
   |+++++++++++++++++++++++++                         | 49% ~35s          
   |+++++++++++++++++++++++++                         | 50% ~34s          
   |++++++++++++++++++++++++++                        | 51% ~33s          
   |+++++++++++++++++++++++++++                       | 52% ~33s          
   |+++++++++++++++++++++++++++                       | 54% ~32s          
   |++++++++++++++++++++++++++++                      | 55% ~31s          
   |++++++++++++++++++++++++++++                      | 56% ~31s          
   |+++++++++++++++++++++++++++++                     | 57% ~30s          
   |++++++++++++++++++++++++++++++                    | 58% ~29s          
   |++++++++++++++++++++++++++++++                    | 60% ~28s          
   |+++++++++++++++++++++++++++++++                   | 61% ~27s          
   |+++++++++++++++++++++++++++++++                   | 62% ~26s          
   |++++++++++++++++++++++++++++++++                  | 63% ~26s          
   |+++++++++++++++++++++++++++++++++                 | 64% ~25s          
   |+++++++++++++++++++++++++++++++++                 | 65% ~24s          
   |++++++++++++++++++++++++++++++++++                | 67% ~23s          
   |++++++++++++++++++++++++++++++++++                | 68% ~22s          
   |+++++++++++++++++++++++++++++++++++               | 69% ~21s          
   |++++++++++++++++++++++++++++++++++++              | 70% ~21s          
   |++++++++++++++++++++++++++++++++++++              | 71% ~20s          
   |+++++++++++++++++++++++++++++++++++++             | 73% ~19s          
   |+++++++++++++++++++++++++++++++++++++             | 74% ~18s          
   |++++++++++++++++++++++++++++++++++++++            | 75% ~17s          
   |+++++++++++++++++++++++++++++++++++++++           | 76% ~17s          
   |+++++++++++++++++++++++++++++++++++++++           | 77% ~16s          
   |++++++++++++++++++++++++++++++++++++++++          | 79% ~15s          
   |++++++++++++++++++++++++++++++++++++++++          | 80% ~14s          
   |+++++++++++++++++++++++++++++++++++++++++         | 81% ~13s          
   |++++++++++++++++++++++++++++++++++++++++++        | 82% ~12s          
   |++++++++++++++++++++++++++++++++++++++++++        | 83% ~12s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~11s          
   |+++++++++++++++++++++++++++++++++++++++++++       | 86% ~10s          
   |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~09s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 88% ~08s          
   |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~07s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 90% ~07s          
   |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~06s          
   |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~05s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 94% ~04s          
   |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~03s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 96% ~02s          
   |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~02s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~01s          
   |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 11s

   |                                                  | 0 % ~calculating  
   |+                                                 | 1 % ~03m 39s      
   |++                                                | 2 % ~03m 36s      
   |++                                                | 3 % ~03m 35s      
   |+++                                               | 4 % ~03m 32s      
   |+++                                               | 5 % ~03m 30s      
   |++++                                              | 6 % ~03m 28s      
   |++++                                              | 7 % ~03m 25s      
   |+++++                                             | 8 % ~03m 23s      
   |+++++                                             | 9 % ~03m 20s      
   |++++++                                            | 10% ~03m 18s      
   |++++++                                            | 11% ~03m 17s      
   |+++++++                                           | 12% ~03m 15s      
   |+++++++                                           | 14% ~03m 13s      
   |++++++++                                          | 15% ~03m 13s      
   |++++++++                                          | 16% ~03m 14s      
   |+++++++++                                         | 17% ~03m 12s      
   |+++++++++                                         | 18% ~03m 09s      
   |++++++++++                                        | 19% ~03m 07s      
   |++++++++++                                        | 20% ~03m 04s      
   |+++++++++++                                       | 21% ~03m 02s      
   |+++++++++++                                       | 22% ~02m 59s      
   |++++++++++++                                      | 23% ~02m 57s      
   |++++++++++++                                      | 24% ~02m 54s      
   |+++++++++++++                                     | 25% ~02m 51s      
   |++++++++++++++                                    | 26% ~02m 49s      
   |++++++++++++++                                    | 27% ~02m 46s      
   |+++++++++++++++                                   | 28% ~02m 44s      
   |+++++++++++++++                                   | 29% ~02m 42s      
   |++++++++++++++++                                  | 30% ~02m 40s      
   |++++++++++++++++                                  | 31% ~02m 37s      
   |+++++++++++++++++                                 | 32% ~02m 35s      
   |+++++++++++++++++                                 | 33% ~02m 32s      
   |++++++++++++++++++                                | 34% ~02m 30s      
   |++++++++++++++++++                                | 35% ~02m 27s      
   |+++++++++++++++++++                               | 36% ~02m 25s      
   |+++++++++++++++++++                               | 38% ~02m 22s      
   |++++++++++++++++++++                              | 39% ~02m 21s      
   |++++++++++++++++++++                              | 40% ~02m 19s      
   |+++++++++++++++++++++                             | 41% ~02m 16s      
   |+++++++++++++++++++++                             | 42% ~02m 14s      
   |++++++++++++++++++++++                            | 43% ~02m 11s      
   |++++++++++++++++++++++                            | 44% ~02m 09s      
   |+++++++++++++++++++++++                           | 45% ~02m 07s      
   |+++++++++++++++++++++++                           | 46% ~02m 04s      
   |++++++++++++++++++++++++                          | 47% ~02m 04s      
   |++++++++++++++++++++++++                          | 48% ~02m 01s      
   |+++++++++++++++++++++++++                         | 49% ~01m 59s      
   |+++++++++++++++++++++++++                         | 50% ~01m 56s      
   |++++++++++++++++++++++++++                        | 51% ~01m 54s      
   |+++++++++++++++++++++++++++                       | 52% ~01m 52s      
   |+++++++++++++++++++++++++++                       | 53% ~01m 49s      
   |++++++++++++++++++++++++++++                      | 54% ~01m 46s      
   |++++++++++++++++++++++++++++                      | 55% ~01m 44s      
   |+++++++++++++++++++++++++++++                     | 56% ~01m 41s      
   |+++++++++++++++++++++++++++++                     | 57% ~01m 39s      
   |++++++++++++++++++++++++++++++                    | 58% ~01m 36s      
   |++++++++++++++++++++++++++++++                    | 59% ~01m 34s      
   |+++++++++++++++++++++++++++++++                   | 60% ~01m 31s      
   |+++++++++++++++++++++++++++++++                   | 61% ~01m 29s      
   |++++++++++++++++++++++++++++++++                  | 62% ~01m 27s      
   |++++++++++++++++++++++++++++++++                  | 64% ~01m 24s      
   |+++++++++++++++++++++++++++++++++                 | 65% ~01m 22s      
   |+++++++++++++++++++++++++++++++++                 | 66% ~01m 19s      
   |++++++++++++++++++++++++++++++++++                | 67% ~01m 17s      
   |++++++++++++++++++++++++++++++++++                | 68% ~01m 14s      
   |+++++++++++++++++++++++++++++++++++               | 69% ~01m 12s      
   |+++++++++++++++++++++++++++++++++++               | 70% ~01m 09s      
   |++++++++++++++++++++++++++++++++++++              | 71% ~01m 07s      
   |++++++++++++++++++++++++++++++++++++              | 72% ~01m 05s      
   |+++++++++++++++++++++++++++++++++++++             | 73% ~01m 02s      
   |+++++++++++++++++++++++++++++++++++++             | 74% ~60s          
   |++++++++++++++++++++++++++++++++++++++            | 75% ~58s          
   |+++++++++++++++++++++++++++++++++++++++           | 76% ~55s          
   |+++++++++++++++++++++++++++++++++++++++           | 77% ~53s          
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Display the top markers you computed above.

tiss.markers %>% group_by(cluster) %>% top_n(20, avg_logFC)

Generate an expression heatmap for given cells and genes. In this case, we are plotting the top 20 markers (or all markers if less than 20) for each cluster.

Assigning cell type identity to clusters

At a coarse level, we can use canonical markers to match the unbiased clustering to known cell types: Cluster 0: basal cell of epidermis (CL:0002187) Cluster 1: differentiated keratinocytes (CL:0000312) Cluster 2: proliferating basal cell of epidermis (CL:0002187) Clutser 3: basal cell of epidermis (CL:0002187) Cluster 4: suprabasal differentiating keratinocytes (CL:0000312) Cluster 5: basal cell of epidermis (CL:0002187) Cluster 6: suprabasal differentiated keratinocytes (CL:0000312) Cluster 7: basal cell of epidermis (CL:0002187) Clutser 8: suprabasal differentiated keratinocytes (CL:0000312) Cluster 9: filiform differentiated keratinocytes (CL:0000312) Cluster 10: Langerhans cells (CL:0000453)

# stash current cluster IDs
tiss <- StashIdent(object = tiss, save.name = "cluster.ids")
# enumerate current cluster IDs and the labels for them
cluster.ids <- c(0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)
free_annotation <- c(
 "basal_cells", 
 "differentiated", 
 "proliferating", 
 "basal_cells", 
 "suprabasal_differentiating", 
 "basal_cells", 
 "suprabasal_differentiated", 
 "basal_cells", 
 "suprabasal_differentiated", 
 "filiform_differentiated", 
 "Langerhans cells"
 )
cell_ontology_class <- c(
"basal cell of epidermis", 
"keratinocyte", 
"basal cell of epidermis",
"basal cell of epidermis",
"keratinocyte",
"basal cell of epidermis",
"keratinocyte",
"basal cell of epidermis",
"keratinocyte",
"keratinocyte",
 "Langerhans cells"
 )
cell_ontology_id <- c(
"CL:0002187",
"CL:0000312", 
"CL:0002187", 
"CL:0002187", 
"CL:0000312", 
"CL:0002187", 
"CL:0000312", 
"CL:0002187", 
"CL:0000312", 
"CL:0000312", 
"CL:0000453"
)
tiss@meta.data[,'free_annotation'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = free_annotation)
tiss@meta.data[,'cell_ontology_class'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_class)
tiss@meta.data[,'cell_ontology_id'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_id)
tiss@meta.data[tiss@cell.names,'free_annotation'] <- as.character(tiss@meta.data$free_annotation)
tiss@meta.data[tiss@cell.names,'cell_ontology_class'] <- as.character(tiss@meta.data$cell_ontology_class)
tiss@meta.data[tiss@cell.names,'cell_ontology_id'] <- as.character(tiss@meta.data$cell_ontology_id)
TSNEPlot(object = tiss, do.label = TRUE, pt.size = 0.5, group.by='cell_ontology_class')

Supplementary Figures

Fig. suppTongue1. tSNE based on free annotation

Fig. suppTongue2. Expression of genes used for annotation of the clusters. Feature plot from both platforms on: Top2a, Cdc20, Mki67, Krt15, Krt5, Krt14, Sbsn, Krt10, Krt4, Krt13, Krt36, Krt84, Hoxc13

Fig. suppTongue3. Expression of genes known to be expressed in taste receptor cells. Violin plot from both platforms on: Krt8, Tas1r1, Tas1r2, Tas1r3, Ncam1, Car4

Checking for batch effects

Color by metadata, like plate barcode, to check for batch effects.

TSNEPlot(object = tiss, do.return = TRUE, group.by = "channel")

TSNEPlot(object = tiss, do.return = TRUE, group.by = "mouse.sex")

Print a table showing the count of cells in each identity category from each plate.

table(as.character(tiss@ident), as.character(tiss@meta.data$channel))
    
     10X_P4_0 10X_P4_1 10X_P7_10
  0       655      521       213
  1       505      296       294
  10       16       10        12
  2       315      317       447
  3       369      348       152
  4       394      271       150
  5       227      112       263
  6       229      252       120
  7       160      221       104
  8       153       65       148
  9        78       75        46

Save the Robject for later

When you save the annotated tissue, please give it a name.

filename = here('00_data_ingest', '04_tissue_robj_generated', 
          paste0("droplet", tissue_of_interest, "_seurat_tiss.Robj"))
print(filename)
[1] "/Users/Jintonic/Documents/GitHub/tabula-muris/00_data_ingest/04_tissue_robj_generated/dropletTongue_seurat_tiss.Robj"
save(tiss, file=filename)
# To reload a saved object
filename = here('00_data_ingest', '04_tissue_robj_generated', 
                      paste0("droplet", tissue_of_interest, "_seurat_tiss.Robj"))
 load(file=filename)

Export the final metadata

So that Biohub can easily combine all your cell_ontology_classs, please export them as a simple csv.

head(tiss@meta.data)
filename = here('00_data_ingest', '03_tissue_annotation_csv', 
          paste0(tissue_of_interest, "_droplet_cell_ontology_class.csv"))
write.csv(FetchData(tiss, c('channel','cell_ontology_class','cell_ontology_id', 'free_annotation', 'tSNE_1', 'tSNE_2')), file=filename)
---
 title: "Tongue Droplet Notebook"
 output: html_notebook
---

Specify the tissue of interest, run the boilerplate code which sets up the functions and environment, load the tissue object.

```{r}
tissue_of_interest = "Tongue"
library(here)
source(here("00_data_ingest", "02_tissue_analysis_rmd", "boilerplate.R"))
tiss = load_tissue_droplet(tissue_of_interest)
```

```{r, echo=FALSE, fig.height=16, fig.width=8}
#PCHeatmap(object = tiss, pc.use = 1:3, cells.use = 500, do.balanced = TRUE, label.columns = FALSE, num.genes = 8)
PCHeatmap(object = tiss, pc.use = 1:10, cells.use = 500, do.balanced = TRUE, 
    label.columns = FALSE, use.full = FALSE)
```

Later on (in FindClusters and TSNE) you will pick a number of principal components to use. This has the effect of keeping the major directions of variation in the data and, ideally, supressing noise. There is no correct answer to the number to use, but a decent rule of thumb is to go until the plot plateaus.

```{r}
PCElbowPlot(object = tiss)
```

Choose the number of principal components to use.
```{r}
# Set number of principal components. 
n.pcs = 14
```


The clustering is performed based on a nearest neighbors graph. Cells that have similar expression will be joined together. The Louvain algorithm looks for groups of cells with high modularity--more connections within the group than between groups. The resolution parameter determines the scale...higher resolution will give more clusters, lower resolution will give fewer.

For the top-level clustering, aim to under-cluster instead of over-cluster. It will be easy to subset groups and further analyze them below.

```{r}
# Set resolution 
res.used <- 0.4

tiss <- FindClusters(object = tiss, reduction.type = "pca", dims.use = 1:n.pcs, 
    resolution = res.used, print.output = 0, save.SNN = TRUE, force.recalc = TRUE)
```


To visualize 
```{r}
# If cells are too spread out, you can raise the perplexity. If you have few cells, try a lower perplexity (but never less than 10).
tiss <- RunTSNE(object = tiss, dims.use = 1:n.pcs, seed.use = 20, perplexity=35, dim.embed = 2)
TSNEPlot(object = tiss, do.label = T, pt.size = 1.2, label.size = 4)
```


Check expression of genes of interset.

```{r, echo=FALSE, fig.height=16, fig.width=10}
genes_to_check = c('Top2a', 'Cdc20', 'Mki67', 'Krt15', 'Krt5', 'Krt14', 'Sbsn', 'Krt10', 'Krt4', 'Krt13', 'Krt36', 'Krt84', 'Hoxc13', 'Krt8', 'Tas1r1', 'Tas1r2', 'Tas1r3', 'Ncam1', 'Car4')
FeaturePlot(tiss, genes_to_check, pt.size = 1, nCol = 3)
```

Dotplots let you see the intensity of exppression and the fraction of cells expressing for each of your genes of interest.

```{r, echo=FALSE, fig.height=4, fig.width=20}
# To change the y-axis to show raw counts, add use.raw = T.
DotPlot(tiss, genes_to_check, plot.legend = T)
```


```{r, echo=FALSE, fig.height=20, fig.width=20}
# To change the y-axis to show raw counts, add use.raw = T.
VlnPlot(tiss, genes_to_check, y.log = TRUE)
```


How big are the clusters?
```{r}
table(tiss@ident)
```

```{r}
tiss1=BuildClusterTree(tiss)
```

```{r}
#cluster1.markers <- FindMarkers(object = tiss, ident.1 = 1, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster2.markers <- FindMarkers(object = tiss, ident.1 = 2, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster3.markers <- FindMarkers(object = tiss, ident.1 = 3, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster4.markers <- FindMarkers(object = tiss, ident.1 = 4, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster5.markers <- FindMarkers(object = tiss, ident.1 = 5, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
cluster6.markers <- FindMarkers(object = tiss, ident.1 = 6, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster7.markers <- FindMarkers(object = tiss, ident.1 = 7, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster8.markers <- FindMarkers(object = tiss, ident.1 = 8, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster9.markers <- FindMarkers(object = tiss, ident.1 = 9, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
#cluster0.markers <- FindMarkers(object = tiss, ident.1 = 0, min.pct = 0.25, only.pos = TRUE, thresh.use = 0.25)
```

Which markers identify a specific cluster?
```{r}
print(x = head(x = cluster6.markers, n = 30))
```


You can also compute all markers for all clusters at once. This may take some time.
```{r}
tiss.markers <- FindAllMarkers(object = tiss, only.pos = TRUE, min.pct = 0.25, thresh.use = 0.25)
```

Display the top markers you computed above.
```{r}
tiss.markers %>% group_by(cluster) %>% top_n(20, avg_logFC)
```


Generate an expression heatmap for given cells and genes. In this case, we are plotting the top 20 markers (or all markers if less than 20) for each cluster.
```{r, echo=FALSE, fig.height=50, fig.width=35}
#top10 <- tiss.markers %>% group_by(cluster) %>% top_n(10, avg_logFC)
top20 <- tiss.markers %>% group_by(cluster) %>% top_n(20, avg_logFC)
# setting slim.col.label to TRUE will print just the cluster IDS instead of
# every cell name
DoHeatmap(tiss, genes.use = top20$gene, slim.col.label = TRUE, remove.key = TRUE, cex.row = 20, group.cex = 20 )
```



## Assigning cell type identity to clusters

At a coarse level, we can use canonical markers to match the unbiased clustering to known cell types:
Cluster 0: basal cell of epidermis (CL:0002187)
Cluster 1: differentiated keratinocytes (CL:0000312)
Cluster 2: proliferating basal cell of epidermis (CL:0002187)
Clutser 3: basal cell of epidermis (CL:0002187) 
Cluster 4: suprabasal differentiating keratinocytes  (CL:0000312)
Cluster 5: basal cell of epidermis (CL:0002187) 
Cluster 6: suprabasal differentiated keratinocytes (CL:0000312)
Cluster 7: basal cell of epidermis (CL:0002187)
Clutser 8: suprabasal differentiated keratinocytes (CL:0000312)
Cluster 9: filiform differentiated keratinocytes (CL:0000312)
Cluster 10: Langerhans cells (CL:0000453)
```{r}
# stash current cluster IDs
tiss <- StashIdent(object = tiss, save.name = "cluster.ids")

# enumerate current cluster IDs and the labels for them
cluster.ids <- c(0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

free_annotation <- c(
 "basal_cells", 
 "differentiated", 
 "proliferating", 
 "basal_cells", 
 "suprabasal_differentiating", 
 "basal_cells", 
 "suprabasal_differentiated", 
 "basal_cells", 
 "suprabasal_differentiated", 
 "filiform_differentiated", 
 "Langerhans cells"
 )
cell_ontology_class <- c(
"basal cell of epidermis", 
"keratinocyte", 
"basal cell of epidermis",
"basal cell of epidermis",
"keratinocyte",
"basal cell of epidermis",
"keratinocyte",
"basal cell of epidermis",
"keratinocyte",
"keratinocyte",
 "Langerhans cells"
 )

cell_ontology_id <- c(
"CL:0002187",
"CL:0000312", 
"CL:0002187", 
"CL:0002187", 
"CL:0000312", 
"CL:0002187", 
"CL:0000312", 
"CL:0002187", 
"CL:0000312", 
"CL:0000312", 
"CL:0000453"
)

tiss@meta.data[,'free_annotation'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = free_annotation)
tiss@meta.data[,'cell_ontology_class'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_class)
tiss@meta.data[,'cell_ontology_id'] <- plyr::mapvalues(x = tiss@ident, from = cluster.ids, to = cell_ontology_id)

tiss@meta.data[tiss@cell.names,'free_annotation'] <- as.character(tiss@meta.data$free_annotation)
tiss@meta.data[tiss@cell.names,'cell_ontology_class'] <- as.character(tiss@meta.data$cell_ontology_class)
tiss@meta.data[tiss@cell.names,'cell_ontology_id'] <- as.character(tiss@meta.data$cell_ontology_id)

TSNEPlot(object = tiss, do.label = TRUE, pt.size = 0.5, group.by='cell_ontology_class')
```

## Supplementary Figures 

Fig. suppTongue1. tSNE based on free annotation

Fig. suppTongue2. Expression of genes used for annotation of the clusters. Feature plot from both platforms on: Top2a, Cdc20, Mki67, Krt15, Krt5, Krt14, Sbsn, Krt10, Krt4, Krt13, Krt36, Krt84, Hoxc13

Fig. suppTongue3.  Expression of genes known to be expressed in taste receptor cells.  Violin plot from both platforms on: Krt8, Tas1r1, Tas1r2, Tas1r3, Ncam1, Car4



## Checking for batch effects

Color by metadata, like plate barcode, to check for batch effects.
```{r}
TSNEPlot(object = tiss, do.return = TRUE, group.by = "channel")
```

```{r}
TSNEPlot(object = tiss, do.return = TRUE, group.by = "mouse.sex")
```

Print a table showing the count of cells in each identity category from each plate.

```{r}
table(as.character(tiss@ident), as.character(tiss@meta.data$channel))
```

# Save the Robject for later
When you save the annotated tissue, please give it a name.

```{r}
filename = here('00_data_ingest', '04_tissue_robj_generated', 
		  paste0("droplet", tissue_of_interest, "_seurat_tiss.Robj"))
print(filename)
save(tiss, file=filename)
```

```{r}
# To reload a saved object
filename = here('00_data_ingest', '04_tissue_robj_generated', 
                      paste0("droplet", tissue_of_interest, "_seurat_tiss.Robj"))
 load(file=filename)
```


# Export the final metadata

So that Biohub can easily combine all your cell_ontology_classs, please export them as a simple csv.

```{r}
head(tiss@meta.data)
```


```{r}
filename = here('00_data_ingest', '03_tissue_annotation_csv', 
		  paste0(tissue_of_interest, "_droplet_cell_ontology_class.csv"))
write.csv(FetchData(tiss, c('channel','cell_ontology_class','cell_ontology_id', 'free_annotation', 'tSNE_1', 'tSNE_2')), file=filename)
```
